The invention relates to methods for enriching nucleic acids from rare cells relative to nucleic acids from non-rare cells.
Cell filtration for the separation of cancer cells using a porous matrix is used to sort cells by size and, in most instances, such filtration methods allow for the extraction of cells following separation. Both microfluidic post and microfluidic membrane methods are used in these filtration approaches. Rare cells contain only small amounts of nucleic acids and these amounts are well below what can be detected by methods applied prior to separation of rare cells from non-rare cells. Currently, in order to analyze nucleic acids in a sample containing a few rare cells (1 to 1000 cells/10 mL), the nucleic acids must be concentrated. However, this does not increase the purity of the nucleic acids, that is, the ratio of nucleic acids associated with a disease state (disease-associated nucleic acids) to nucleic acids not associated with a disease state (non-disease-associated nucleic acids). Impurities are due to nucleic acids from non-diseased cells, which occur from freely circulating nucleic acids and to nucleic acids from non-rare cells. These impurities make the analysis of nucleic acids from diseased cells insensitive and prone to false results. Therefore, even though methods are known for enhancing an amount of rare cells over non-rare cells, enough of non-disease-associated nucleic acids remain in a sample such that the amount of disease-associated nucleic acids cannot be accurately determined because of the non-disease-associated nucleic acids.
Furthermore, cell filtration typically requires fixation of cells for high recovery of diseased cells. Without fixation, less than 40% recovery of diseased cells is realized whereas with fixation, the recovery is greater than 90%. Fixation is also required for sample stability. However, fixation causes problems as nucleic acids are usually heavily fragmented and chemically modified by a fixation agent such as, for example, formaldehyde. Although formaldehyde modification cannot be detected in standard quality control assays such as, for example, gel electrophoresis, it does strongly interfere with analysis of nucleic acids. While nucleic acid isolation and purification methods can be optimized to reverse as much formaldehyde modification as possible without further RNA degradation, RNA purified from fixed samples is not a good candidate for downstream applications that require full-length RNA such as, for example, polymerase chain reaction methods.
Nucleic acids from rare cells can be found inside rare cells or as circulating nucleic acids free of the cells. Analysis of both cellular and cell free nucleic acids are important. Cellular nucleic acids within rare cells in circulation, such as from circulating tumor cells, are found in patients with cancer. These cells are intact, alive and can often replicate. However the nucleic acids quantities from these cells are of extremely low concentration and in the range of attogram per tube of blood (7-10 mL). This is due the small numbers of these rare cells per tube of blood, for example 1 to 300 circulating tumor cells. However, these cells can survive in blood and cause disease to spread when the cells enter into the tissues of the body. The latency time that these cells can survive in blood is extremely long and on the order of years. Therefore, it is important to know if these diseased rare cells are present and to analyze their nucleic acids to determine whether such cells will adapt and survived the stress of blood with potential to enter tissue or cause a treatment resistant disease.
Cell free nucleic acids arise mainly from rare cells that reside inside tissues, such as rare cells from inside solid cancer tumors and infected tissues. These cell free nucleic acids are released into the circulation in a much higher concentration than nucleic acids from circulating rare cells. The observed range of cell free circulating nucleic acids in blood is between 200 ng to 40 μg per 10 mL of blood in healthy persons. During disease, cell free nucleic acids increased if significant disease is occurring in the tissues. In order to produce nanograms of cell free nucleic acids, billions of rare disease cells must release nucleic acids. Therefore the minimum amount of diseased tissue with rare cells is of significant size, for example a tumor above a critical mass of cm size.
The principle of particle capture is well known and has been used to bind, isolate, separate and concentrate nucleic acids in circulation, such as RNA or DNA (Vogelstein B, Gillespie D., Proc Natl Acad Sci 1979:79:615-19). One of early procedures were developed for removing DNA, involves using glass particles prepared from ground scintillation vials (American Flint Glass Co.). At the time, silica gel and porous glass beads were unsuitable for nucleic acid removal. Recently, silica coated magnetic particles have been found useful to isolate, separate and concentrate nucleic acids, such as RNA or DNA (WO 03/058649, U.S. Pat. Nos. 8,846,897 and 8,703,931). These magnetic particles have a non-porous, ultrathin silica layer and are able to isolate and separate nucleic acids from tissue samples and biological fluids by making use of the silica layer to bind the nucleic acids and the magnetic properties to hold particles during wash steps. The methods are non-selective and capture all nucleic acids whether from rare cells or non-rare cells.
The problem of purity of nucleic acids is exacerbated by methods of isolating nucleic acids. Such methods employ reagents such as, for example, detergents or phenols, which can damage the nucleic acid material. Furthermore, contamination of nucleic acids with other reagents such as organic solvents and other extraction chemicals can affect the integrity of nucleic acid samples. Other integrity problems include degradation, fragmentation, and binding and crosslinking of nucleic acids.
There is, therefore, a need to develop a method of enriching disease-associated nucleic acids over non-disease-associated nucleic acids. The method should improve nucleic acid recovery from rare cells and reduce or eliminate contamination and allow for sensitive and accurate determination of nucleic acids in rare cells.